Characterization of the nar promoter to use as an inducible promoter

Summary The nar promoter of Escherichia coli was characterized, which is maximally induced under anaerobic conditions in the presence of nitrate. The following results were obtained; Expression of -galactosidase was optimal at 1 % of nitrate and was not affected much by molybdate; the amount of -galactosidase per unit volume was maximal when the nar promoter was induced at OD₆₀₀ = 1.7, and when anaerobic condition was made by supplying nitrogen gas. At the optimal condition, the ratio of -galactosidase between before and after induction was approximately 250 and Miller units were approximately 7,500. The results showed that the nar promoter can be used as an inducible promoter.     

Mutagens and the tumor promoter

We studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and saccharin on the frequency of induced mutations of resistance to 6-mercaptopurine and ouabain in Chinese hamster and mouse cells. UV-rays, bovine adenovirus-3 (BAV-3) and 5-bromdeoxyuridine (BrdU) were used as mutagens. In the case of BAV-3 and BrdU, we investigated, apart from the mutagenic effect, the tumor-inducing activity of these mutagens in mice, BrdU proved to have no carcinogenic effect. The data about the influence of TPA on the mutagenic effect of the three different mutagens indicate that TPA increases the frequency of the gene mutations induced by UV-rays and BAV-3. The results of the study of BrdU and TPA combined action revealed the fact that TPA does not increase the mutagenic effect of BrdU. We demonstrated that saccharin also possesses the promoter activity; it increases the mutagenic effect of BAV-3. The results described above lead to the assumption that TPA influence on the mutagenic effect only takes place when carcinogenic mutagens are used.     

Estrogen regulates the IFN-gamma promoter

The greater immune reactivity of females has been attributed in part to the influence of sex steroid hormones, but the underlying mechanisms are unknown. Here we report evidence that expression of the IFN-gamma gene may be subject to direct hormonal control. In a transient expression assay, the sex steroid 17 beta-estradiol markedly increases activity of the IFN-gamma promoter in lymphoid cells that express the appropriate hormone receptor. This effect is mediated by sequences in the 5'-flanking region of the gene, and can augment the effect of T cell-activating agents. Short term exposure to estradiol also increases IFN-gamma mRNA expression in Con A-treated murine spleen cells. Hormonal regulation of this pleiotropic cytokine may account in part for the ability of estrogen to potentiate many types of immune responses, and for the disproportionate susceptibility of females to autoimmune disease.     

An Escherichia coli gene divergently transcribed from a promoter overlapping the metA promoter

The upstream region of the metA gene in Escherichia coli contains two promoters. We have identified by lacZ fusion an additional promoter in this region, and showed that it is transcribed in the opposite orientation from the metA gene. The putative translation product corresponds to a peptide of 147 amino acids – ORF19 by molecular mass. This peptide is probably not essential for growth, as an insertion mutant is viable.     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

Mapping the rubella virus subgenomic promoter

Rubella virus (RUB), the sole member of the Rubivirus genus in the Togaviridae family of positive-strand RNA viruses, synthesizes a single subgenomic (SG) RNA containing sequences from the 3' end of the genomic RNA including the open reading frame (ORF) that encodes the virion proteins. The synthesis of SG RNA is initiated internally on a negative-strand, genome-length template at a site known as the SG promoter (SGP). Mapping the RUB SGP was initiated by using an infectious cDNA vector, dsRobo402/GFP, in which the region containing the SGP was duplicated (K. V. Pugachev, W.-P. Tzeng, and T. K. Frey, J. Virol. 74:10811-10815, 2000). In dsRobo402/GFP, the 5'-proximal nonstructural protein ORF (NS-ORF) is followed by the first SGP (SGP-1), the green fluorescent protein (GFP) gene, the second SGP (SGP-2), and the structural protein ORF. The duplicated SGP, SGP-2, contained nucleotides (nt) -175 to +76 relative to the SG start site, including the 3' 127 nt of the NS-ORF and 47 nt between the NS-ORF and the SG start site. 5' Deletions of SGP-2 to nt -40 (9 nt beyond the 3' end of the NS-ORF) resulted in a wild-type (wt) phenotype in terms of virus replication and RNA synthesis. Deletions beyond this point impaired viability; however, the analysis was complicated by homologous recombination between SGP-1 and SGP-2 that resulted in deletion of the GFP gene and resurrection of viable virus with one SGP. Since the NS-ORF region was not necessary for SGP activity, subsequent mapping was done by using both replicon vectors, RUBrep/GFP and RUBrep/CAT, in which the SP-ORF is replaced with the reporter GFP and chloramphenical acetyltransferase genes, respectively, and the wt infectious clone, Robo402. In the replicon vectors, 5' deletions to nt -26 resulted in the synthesis of SG RNA. In the infectious clone, deletions through nt -28 gave rise to viable virus. A series of short internal deletions confirmed that the region between nt -28 and the SG start site was essential for viability and showed that the repeated UCA triplet at the 5' end of SG RNA was also required. Thus, the minimal SGP maps from nt -26 through the SG start site and appears to extend to at least nt +6, although a larger region is required for the generation of virus with a wt phenotype. Interestingly, while the positioning of the RUB SGP immediately adjacent the SG start site is thus similar to that of members of the genus Alphavirus, the other genus in the Togaviridae family, it does not include a region of nucleotide sequence homology with the alphavirus SGP that is located between nt -48 and nt -23 with respect to the SG start site in the RUB genome.